scholarly journals Functional Roles of Nonconserved Structural Segments in CFTR's NH2-terminal Nucleotide Binding Domain

2004 ◽  
Vol 125 (1) ◽  
pp. 43-55 ◽  
Author(s):  
László Csanády ◽  
Kim W. Chan ◽  
Angus C. Nairn ◽  
David C. Gadsby
Keyword(s):  
Cystic Fibrosis ◽  
Conformational Changes ◽  
Single Channel ◽  
Channel Gating ◽  
Essential Element ◽  
Nucleotide Binding ◽  
Transmembrane Conductance Regulator ◽  
Functional Roles ◽  
Binding Domains ◽  
Excised Patches

The cystic fibrosis transmembrane conductance regulator (CFTR), encoded by the gene mutated in cystic fibrosis patients, belongs to the family of ATP-binding cassette (ABC) proteins, but, unlike other members, functions as a chloride channel. CFTR is activated by protein kinase A (PKA)-mediated phosphorylation of multiple sites in its regulatory domain, and gated by binding and hydrolysis of ATP at its two nucleotide binding domains (NBD1, NBD2). The recent crystal structure of NBD1 from mouse CFTR (Lewis, H.A., S.G. Buchanan, S.K. Burley, K. Conners, M. Dickey, M. Dorwart, R. Fowler, X. Gao, W.B. Guggino, W.A. Hendrickson, et al. 2004. EMBO J. 23:282–293) identified two regions absent from structures of all other NBDs determined so far, a “regulatory insertion” (residues 404–435) and a “regulatory extension” (residues 639–670), both positioned to impede formation of the putative NBD1–NBD2 dimer anticipated to occur during channel gating; as both segments appeared highly mobile and both contained consensus PKA sites (serine 422, and serines 660 and 670, respectively), it was suggested that their phosphorylation-linked conformational changes might underlie CFTR channel regulation. To test that suggestion, we coexpressed in Xenopus oocytes CFTR residues 1–414 with residues 433–1480, or residues 1–633 with 668–1480, to yield split CFTR channels (called 414+433 and 633+668) that lack most of the insertion, or extension, respectively. In excised patches, regulation of the resulting CFTR channels by PKA and by ATP was largely normal. Both 414+433 channels and 633+668 channels, as well as 633(S422A)+668 channels (lacking both the extension and the sole PKA consensus site in the insertion), were all shut during exposure to MgATP before addition of PKA, but activated like wild type (WT) upon phosphorylation; this indicates that inhibitory regulation of nonphosphorylated WT channels depends upon neither segment. Detailed kinetic analysis of 414+433 channels revealed intact ATP dependence of single-channel gating kinetics, but slightly shortened open bursts and faster closing from the locked-open state (elicited by ATP plus pyrophosphate or ATP plus AMPPNP). In contrast, 633+668 channel function was indistinguishable from WT at both macroscopic and microscopic levels. We conclude that neither nonconserved segment is an essential element of PKA- or nucleotide-dependent regulation.

scholarly journals Perturbation of the Pore of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Inhibits Its ATPase Activity

2000 ◽  
Vol 276 (15) ◽  
pp. 11575-11581 ◽  
Author(s):  
Ilana Kogan ◽  
Mohabir Ramjeesingh ◽  
Ling-Jun Huan ◽  
Yanchun Wang ◽  
Christine E. Bear
Keyword(s):  
Cystic Fibrosis ◽  
Atpase Activity ◽  
Cross Talk ◽  
Nucleotide Binding ◽  
Cystic Fibrosis Gene ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Binding Domains ◽  
Cystic Fibrosis Transmembrane

Mutations in the cystic fibrosis gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR) lead to altered chloride (Cl−) flux in affected epithelial tissues. CFTR is a Cl−channel that is regulated by phosphorylation, nucleotide binding, and hydrolysis. However, the molecular basis for the functional regulation of wild type and mutant CFTR remains poorly understood. CFTR possesses two nucleotide binding domains, a phosphorylation-dependent regulatory domain, and two transmembrane domains that comprise the pore through which Cl−permeates. Mutations of residues lining the channel pore (e.g.R347D) are typically thought to cause disease by altering the interaction of Cl−with the pore. However, in the present study we show that the R347D mutation and diphenylamine-2-carboxylate (an open pore inhibitor) also inhibit CFTR ATPase activity, revealing a novel mechanism for cross-talk from the pore to the catalytic domains. In both cases, the reduction in ATPase correlates with a decrease in nucleotide turnover rather than affinity. Finally, we demonstrate that glutathione (GSH) inhibits CFTR ATPase and that this inhibition is altered in the CFTR-R347D variant. These findings suggest that cross-talk between the pore and nucleotide binding domains of CFTR may be important in thein vivoregulation of CFTR in health and disease.

scholarly journals Curcumin Opens Cystic Fibrosis Transmembrane Conductance Regulator Channels by a Novel Mechanism That Requires neither ATP Binding nor Dimerization of the Nucleotide-binding Domains

2006 ◽  
Vol 282 (7) ◽  
pp. 4533-4544 ◽  
Author(s):  
Wei Wang ◽  
Karen Bernard ◽  
Ge Li ◽  
Kevin L. Kirk
Keyword(s):  
Cystic Fibrosis ◽  
Nucleotide Binding ◽  
Salt Transport ◽  
Atp Binding ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Binding Domains ◽  
Channel Opening ◽  
Cystic Fibrosis Transmembrane ◽  
R Domain

Cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels are essential mediators of salt transport across epithelia. Channel opening normally requires ATP binding to both nucleotide-binding domains (NBDs), probable dimerization of the two NBDs, and phosphorylation of the R domain. How phosphorylation controls channel gating is unknown. Loss-of-function mutations in the CFTR gene cause cystic fibrosis; thus, there is considerable interest in compounds that improve mutant CFTR function. Here we investigated the mechanism by which CFTR is activated by curcumin, a natural compound found in turmeric. Curcumin opened CFTR channels by a novel mechanism that required neither ATP nor the second nucleotide-binding domain (NBD2). Consequently, this compound potently activated CF mutant channels that are defective for the normal ATP-dependent mode of gating (e.g. G551D and W1282X), including channels that lack NBD2. The stimulation of NBD2 deletion mutants by curcumin was strongly inhibited by ATP binding to NBD1, which implicates NBD1 as a plausible activation site. Curcumin activation became irreversible during prolonged exposure to this compound following which persistently activated channels gated dynamically in the absence of any agonist. Although CFTR activation by curcumin required neither ATP binding nor heterodimerization of the two NBDs, it was strongly dependent on prior channel phosphorylation by protein kinase A. Curcumin is a useful functional probe of CFTR gating that opens mutant channels by circumventing the normal requirements for ATP binding and NBD heterodimerization. The phosphorylation dependence of curcumin activation indicates that the R domain can modulate channel opening without affecting ATP binding to the NBDs or their heterodimerization.

scholarly journals ATP hydrolysis-driven gating in cystic fibrosis transmembrane conductance regulator

2008 ◽  
Vol 364 (1514) ◽  
pp. 247-255 ◽  
Author(s):  
Daniella Muallem ◽  
Paola Vergani
Keyword(s):  
Cystic Fibrosis ◽  
Conformational Changes ◽  
Atp Hydrolysis ◽  
Atp Binding ◽  
Functional Difference ◽  
Transmembrane Conductance Regulator ◽  
Cellular Functions ◽  
Transmembrane Conductance ◽  
Binding Domains ◽  
Cystic Fibrosis Transmembrane

Proteins belonging to the ATP-binding cassette superfamily couple ATP binding and hydrolysis at conserved nucleotide-binding domains (NBDs) to diverse cellular functions. Most superfamily members are transporters, while cystic fibrosis transmembrane conductance regulator (CFTR), alone, is an ion channel. Despite this functional difference, recent results have suggested that CFTR shares a common molecular mechanism with other members. ATP binds to partial binding sites on the surface of the two NBDs, which then associate to form a NBD dimer, with complete composite catalytic sites now buried at the interface. ATP hydrolysis and γ-phosphate dissociation, with the loss of molecular contacts linking the two sides of the composite site, trigger dimer dissociation. The conformational signals generated by NBD dimer formation and dissociation are transmitted to the transmembrane domains where, in transporters, they drive the cycle of conformational changes that translocate the substrate across the membrane; in CFTR, they result in opening and closing (gating) of the ion-permeation pathway.

Sign in / Sign up

Export Citation Format

Share Document